Thyrotropin-releasing hormone (TRH) has been shown to regulate prolactin release and synthesis independently by two separate hormone receptor interactions, although it has not yet been shown whether these interactions are caused by two physically distinct sites, or by two interactions with one site. In order to see if this separation of prolactin release and synthesis occurs with other compounds, I plan to see if bromocriptine, which decreases prolactin release, and enkephalin, which increases prolactin release, also separately regulate prolactin release and synthesis. If this is the case I plan to determine if the two processes are mediated by one receptor or more than one by the use of antagonists available for these compounds. Three in vitro systems will be used to study prolactin production: (1) clonal strains of rat pituitary tumor cells known as GH-cells, (2) dispersed primary cultures of normal rat pituitary glands, and (3) dispersed primary cultures of pig pituitary glands. I will investigate analogs of TRH, which, if only one receptor exists, will have the property of antagonizing release since they bind but do not cause release. I will investigate analogs of TRH which may distinguish between prolactin and thyrotropin release. I plan to see if bromocriptine and enkephalin affect prolactin synthesis as well as prolactin release, and if these two processes have the same analog specificity for each of these two compounds. I will see if one process can be preferentially inhibited by an antagonist of bromocriptine or enkephalin. This will provide functional evidence for one or two receptors. Binding can also be studied directly. The specific binding of (3H) dihydroergocriptine to pig pituitary membranes can be measured; I plan to characterize the binding of dihydroergocriptine and investigate the binding potency of various analogs to see how this corresponds to the biological activities of prolactin release and synthesis. The binding of (3H) enkephalin and morphine analogs will also be studied and correlated with biological activities.